The long-term goal of our work is to develop bacteriophage as a paradigm-shifting approach for treating infections caused by multidrug-resistant (MDR) Gram-negative pathogens. As a first step towards this new therapeutic approach, we will explore the use of bacteriophage in the treatment of catheter-associated urinary tract infection (CAUTI), caused by MDR Pseudomonas aeruginosa (Psa). Psa is the second most commonly identified causative organism in CAUTI, and clinical outcomes are worse when it is MDR. With the lack of new antibiotic drugs, Psa is increasingly resistant to available antibiotics and some resistant strains are virtually untreatable with currently available drugs. Phage offer an alternative treatment that has numerous advantages over antibiotics, including that they can kill MDR bacteria by a different mechanism than antibiotics; they can target the infecting Psa without harming other intestinal commensals; they are a self-replicating drug and amplify during treatment; and that they are generally regarded as non-toxic compared to antibiotics. Phage are also likely to be excellent adjuvants to traditional antimicrobials, as tey can disrupt biofilms, and reversion to antibiotic sensitivity has been observed in phage-resistant bacteria. In this R21/R33, we propose to develop the first therapeutic phage to treat multi-drug resistant CAUTI infections caused by P. aeruginosa (Psa). We chose Psa CAUTI as a target because the intrinsic resistance of Psa makes infections difficult to treat, setting a high bar in our studies. Psa also forms biofilms on medical devices, further complicating management. In proof of concept experiments, we will test a Psa-specific phage cocktail in a murine model of CAUTI against a human Psa isolate susceptible to the cocktail (R21). We will also use the mouse CAUTI model to test the concept that phage cocktail and antibiotic may be synergistic (R21). We will then use a phage host range expansion protocol developed by our group to generate phage that can kill a broad-spectrum of clinically-relevant Psa isolates (R21) and confirm that (i) the resulting phage are efficacious in vivo and (ii) are efficacious against contemporary Psa clinical isolates from the Texas- Louisiana region and across the U.S. Finally, in the R33 phase of this work, we will define good manufacturing practice (GMP) for the isolation, administration, and storage of our therapeutic phage, thereby paving a path for regulatory approval for clinical use of our phage in humans. The phage development techniques in this proposal may then be applied to other MDR pathogens.